Immunohistochemistry (IHC) refers to the processes of detecting, localizing, and/or quantifying antigens, such as a protein, in a biological sample using antibodies specific to the particular antigens. IHC provides the substantial advantage of identifying exactly where a particular protein is located within the tissue sample. It is also an effective way to examine the tissues themselves. In situ hybridization (ISH) refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on various biological samples, such as tissue (e.g. fresh frozen, formalin fixed, paraffin embedded) and cytological samples. Recognition of the targets can be detected using various labels (e.g., chromogenic, fluorescent, luminescent, radiometric), irrespective of whether the target is a nucleic acid or an antigen.
To robustly detect, locate, and quantify targets in a clinical setting, amplification of the recognition event is desirable as the ability to confidently detect cellular markers of low abundance becomes increasingly important for diagnostic purposes. For example, depositing at the marker's site hundreds or thousands of label molecules in response to a single antigen detection event enhances, through amplification, the ability to detect that recognition event. Adverse events often accompany amplification, such as non-specific signals that are apparent as an increased background signal. An increased background signal may interfere with the clinical analysis by obscuring faint signals that may be associated with low, but clinically significant, expressions.
Despite efforts to restrict labeling to the target sequence of interest in ISH, anomalous non-specific localization of the detection reagent or DNA probe may occur due to a variety of causes, such as inconsistent performance of staining instruments, instability of reagents and loss of specific binding activity or aggregation, inappropriate staining protocols, and contamination of the slide. The same issues may apply to samples stained with chromogens, fluorophores, and/or quantum dots.
For commercial in-vitro diagnostic ISH assays, the staining performance, e.g. stain uniformity, stain intensity, or background staining, must be validated to deliver sensitive and specific staining to the sequences of interest, with a high degree of repeatability. For this reason, specifications that define the various factors that influence acceptable performance of ISH assays must be developed and documented.
Current methods for ISH performance specifications are based primarily on subjective impressions of various factors recorded on a scale that is developed to aid in statistical measurement over a large number of slides. Such subjective scoring is generally achieved through direct inspection of slides through the oculars of a brightfield or fluorescent microscope. Human perception is largely comparative, and the subjective scoring methods in use cannot be defined to precise standards because of the influence of experience, visual acuity, and understanding of the scaling system being used in a particular context.
In current practice, there are at least three subjective measurements that are weighed in determining overall stain quality assessment for ISH: (i) intensity (or contrast) of the stain localizations, (ii) coverage (the percentage of potential available hybridization sites that exhibit signal), and (iii) background (non-specific localization of signal, due to adherence of either the reporter (quantum dot and conjugated antibody) or adherence of the DNA probe itself. Thus, it is difficult to compare slide-to-slide variation and nearly impossible to define concrete specifications for staining performance.
The aim of the present invention is therefore to provide a means for assessing the stain quality in the fields of in-situ hybridization and immunohistochemistry and to provide a means for establishing objective criteria for assessing the stain quality for in-situ hybridization and immunohistochemistry.